The detection of glucose in body fluids, as well as the determination of its concentration therein, is of great importance for diabetic patients who must control their diets so as to regulate their sugar intake and who must frequently be guided in this regard by a regular check on urine glucose. The determination of glucose in urine is also important where large numbers of people are screened to determine the incidence of diabetes among them.
Because early diagnosis and continued control are so important in diabetes, a glucose test, to be of greater value, must be conveniently rapid, simple enough for the technician or patient to learn with ease, accurate enough to serve the clinician or patient, sensitive enough to reflect variations in the patient's condition, and specific for glucose.
Currently there are available sophisticated biochemical systems which can be incorporated into dry, dip-and-read reagent strip devices, used in solution or suspension techniques, or in conjunction with spectrophotometics and other read-out systems.
These strips comprise a plastic strip, having at one end a carrier portion impregnated with an enzymatic testing composition which includes the enzymes glucose oxidase and peroxidase and one or more indicator compounds as the principal active ingredients. Buffering agents may be present to keep the pH of the reactants at the site of reaction at a predetermined pH range. The strip utilizes an enzyme system wherein the glucose is a substrate for glucose oxidase. Glucose is oxidized to gluconic acid with the concomitant formation of hydrogen peroxide. Indicator compounds present undergo color changes in the presence of hydrogen peroxide and peroxidase. Various indicators can be used including "benzidine-type" chromogens, e.g., benzidine, o-tolidine and tetramethylbenzidine and substituted aniline chromogens. A combination of indicators can be utilized.
The glucose enzymatic test strips referred to above enable the assay of glucose levels by measuring the rate of color change which the indicator undergoes, i.e., by a rate reaction. The sample to be analyzed for glucose is contacted with the reagent-incorporated carrier portion by momentarily immersing the carrier portion into the sample or by applying an aliquot of the sample to the carrier portion and measuring the response after a set period of reaction time, by comparing any color formed in the carrier portion with a standard color chart calibrated to various glucose concentrations.
The general principles of chemical reaction kinetics apply to enzyme-catalyzed reactions, but enzyme-catalyzed reactions also show a distinctive feature not usually observed in nonenzymatic reactions, saturation with substrate. The rate equation for reactions catalyzed by enzymes having a single substrate, e.g., glucose, is expressed by an equation known as the Michaelis-Menten equation. Under certain reaction conditions, the Michaelis-Menten equation can be used to derive a value known as the Michaelis-Menten constant (K.sub.M) [See Biochemistry, Lehninger, 2nd Edition, pp. 189-192]. The equation expresses the mathematical relationship between the initial rate of the enzyme-catalyzed reaction and the concentration of the substrate. At high substrate concentrations, the K.sub.M of the glucose oxidase is exceeded and the reaction rate becomes nearly independently of concentration--this means that at such concentrations, it becomes difficult to determine concentrations of glucose based on a rate reaction color change. In the glucose-glucose oxidase system, as the level of glucose present approaches 2 percent, the K.sub.M of glucose oxidase is exceeded, rendering it difficult to determine with accuracy the glucose level of the sample being tested.
Diabetic patients can have glucose levels ranging from 50 mg/dl (0.05%) to 10,000 mg/dl (10%). Because of this wide range, for detection and treatment purposes, it is important to be able to quantitatively determine glucose levels in a range which encompasses about 1/2 up to 10 percent. At present, dip-and-read reagent strips do not enable determination of glucose levels which exceed about 2 percent.
The present invention overcomes this limitation of dip-and-read reagent strips and provides a method of measuring glucose levels of about 1/2 to about 10 percent.